Particles containing a growth factor, and uses thereof

ABSTRACT

The present invention concerns particles containing at least one covalently cross-linked polysaccharide and at least one growth factor, a method of preparation, and uses thereof.

The present invention relates to particles containing a growth factor,and uses thereof, in particular for the controlled spatiotemporaldelivery of said growth factor.

It also relates to a method for preparing said particles.

Therapeutic delivery of growth factors has been attempted in clinicaltrials, for example in cardiovascular disease patients, but has notshown the expected functional benefits. One reason may be that thegrowth factors used to stimulate a response are rapidly degradedfollowing injection into the circulation or in the target organ, andthat the therapy thus is too short-lived to generate a durable result.

Thus, techniques allowing spatiotemporal control over growth factorrelease rates must be taken into consideration when developing noveltherapeutic angiogenesis therapies, for example for cardiovasculardisease patients.

In contrast to bolus delivery of proteins by systemic or localinjections, delivery using slow-release delivery systems showssignificantly improved efficacy by allowing a local and sustainedrelease of growth factors at low doses (Richardson et al. Nat.Biotechnol. 19, 1029 (2001), A. H. Zisch et al. Cardiovasc. Pathol. 12,295-310 (2003)).

Previous therapies based on polymer delivery of growth factors (C.Fischbach & D. J. Mooney Adv. Polym. Sci. 203, 191-221 (2006),Richardson et al. Nat. Biotechnol. 19, 1029 (2001), F-M Chen et al.Biomaterials 31, 6279-6308 (2010)) have used either non-injectablesolid, implantable scaffolds, or injectable “gel like” formulations,resulting in a non-homogenous tissue distribution pattern of the growthfactors and a poor duration of growth factor delivery.

Various particles, prepared from synthetic or natural biodegradablepolymers or from proteins, have been studied for the encapsulation ofgrowth factors.

Many studies deal with the use of microspheres composed of ionicallycross-linked alginate as drug delivery systems for growth factors.

The aim of the present invention is to provide particles containing atleast one growth factor, which allow a slow release of said growthfactor.

The aim of the present invention is to provide particles able to providea potent, localized and prolonged biological effect, such as a cellulargrowth, proliferation, differentiation and/or maturation.

The present invention relates to a particle containing at least onecovalently cross-linked polysaccharide and at least one growth factor.

In the literature, methods for the preparation of particles made ofcovalently cross-linked polysaccharides can be found (U.S. Pat. No.4,780,321, M C Lévy and M C Andry, Int. J. Pharm. 62, 27-35 (1990); R.Hurteaux et al., Eur. J. Pharm. Sci. 24, 187-197, 2005; M. Callewaert etal., Int. J. Pharm. 366, 103-110 (2009)). However, the use of theseparticles as delivery systems for growth factors is neither suggestednor described.

A study performed on calcium alginate microspheres surrounded with apolysaccharide-protein co-cross-linked membrane (M. Callewaert et al.,Int. J. Pharm. 366, 103-110 (2009)) showed that a cationic bioactivepeptide interacted with the internal ionically cross-linked alginatehydrogel, but did not engage interactions with the material constitutingthe polysaccharide-protein co-crosslinked membrane. Thus, the use ofparticles surrounded by a polysaccharide-protein co-crosslinked membranebut lacking the internal ionically cross-linked alginate hydrogel doesnot appear to a skilled man in the art to be adequate for the loading ofgrowth factors.

Surprisingly, it was found that particles loaded with growth factorsaccording to the invention, though lacking the internal ionicallycross-linked alginate hydrogel, were able to release the loaded growthfactors in a controlled and sustained manner.

The particles of the invention are able to provide a slow release ofsaid growth factor and the biological effects are of longer duration ascompared to growth factor delivery with particles without suchcovalently cross-linked polysaccharide.

The particles of the invention are able to provide a release of saidgrowth factors on a period of at least 15 days, and generally on aperiod of approximately 40 days.

The use of particles as vectors for growth factors brings the advantageof dividing the dose into discrete drug delivery systems that can spreadover a local territory to infuse the therapeutic molecules morehomogeneously, providing a more physiological-like stimulation.Moreover, these particles are also intended to protect the bioactivepeptides until they reach their biological target.

Such particles are particularly advantageous for homogenousconcentration gradients of growth factors inside target organs, forexample for therapeutic stimulation of blood vessel growth (i.e.angiogenesis and arteriogenesis), for stimulating wound healing, fortissue regeneration in vivo or for stimulating transplant organgrafting.

Slow release of growth factors thus induces biological effects. Thestabilization of those effects is permitted by the extended duration ofsuch release.

As used herein, the term “particle” refers to an aggregated physicalunit of solid material.

The particles according to the invention are preferably microparticles.

Microparticles are herein understood as particles having a mediandiameter d₅₀ less than 1 000 μm.

As used herein, the term “median diameter d₅₀” refer to the particlediameter so that 50% of the volume of the particles population have asmaller diameter.

The median diameter d₅₀ according to the invention is determined byvirtue of a particle size measurement performed on the suspensionsaccording to the method based on light diffraction.

In a particular embodiment, a particle according to the invention, alsocalled microcapsule, preferably presents a core/shell structure, alsocalled a core/membrane structure. The cross-linked polysaccharide ispreferably comprised at least in the shell of the particle.Advantageously, the shell of the particle is solid and consistsessentially in the cross-linked polysaccharide.

The core of such particles may be solid, liquid or gaseous, but may notbe an ionically cross-linked hydrogel.

The solid core of the particles may consist in various materialparticles, like polymer, metal, mineral.

The gaseous core of the particles may consist in various gases, likeair, nitrogen or argon.

According to one embodiment, the core of the particles is liquid.

In one embodiment, a particle according to the invention presents acore/membrane structure, wherein:

-   -   the membrane comprises at least one covalently cross-linked        polysaccharide and at least one growth factor, and    -   the core is liquid.

According to another embodiment, a particle according to the inventionpresents a core/membrane structure, wherein:

-   -   the membrane consists in a covalently cross-linked        polysaccharide, on which at least one growth factor is adsorbed,        and    -   the core is liquid.

The membrane generally does not comprise ionically cross-linkedpolysaccharide, such as coacervates of polysaccharides.

In another particular embodiment, a particle according to the inventionpresents a matrix structure, formed of a network comprising at least onecovalently cross-linked polysaccharide.

Said particle is also called microsphere. The network of said particlefills the whole volume of the particle. Advantageously, said network issolid. Said particle may contain a liquid filling the pores of saidnetwork. Said particle may also contain gas bubbles or solid particles.

In another embodiment, a particle according to the invention presents amatrix structure, wherein:

-   -   the constitutive network comprises at least one covalently        cross-linked polysaccharide and at least one growth factor        adsorbed on it, and    -   the network entraps an aqueous solution in its pores.

The median diameter d₅₀ of the particles according to the invention ispreferably comprised from 5 μm to 1 000 μm, more preferably from 50 μmto 200 μm, more preferably from 75 μm to 150 μm, and more preferablyfrom 60 μm to 100 μm.

As used herein, the term “polysaccharide” refers to polymericcarbohydrate structures, formed of repeating units joined together byglycosidic bonds. These structures are often linear, but may containvarious degrees of branching. The term “polysaccharide” refers to asingle polysaccharide or a mixture of two or more polysaccharides.

As used herein, the terms “covalently cross-linked polysaccharide” referto a polymer formed of polysaccharide units, linked together by covalentchemical bonds. Such bonds generally bind together two polymer chains oftwo polysaccharide molecules. Alternatively, such bonds can bindtogether the same polysaccharide molecule, thus forming a loop-shapedpattern.

Such covalently cross-linked polysaccharide is usually obtained from apolysaccharide, or a mixture of two or more different polysaccharides,treated in the presence of a cross-linking agent or using another methodleading to the formation of covalent cross-links between thepolysaccharide molecules.

A method for the preparation of said covalently cross-linkedpolysaccharides will be further described.

The polysaccharide of the particle according to the invention ispreferably chosen from the group consisting of gum Arabic, xanthan gum,gellan gum, acacia gum, tragacanth gum, guar gum, carob gum, karaya gum,alginic acid and derivatives thereof, alginic salts, alginic esters likepropylene glycol alginate, pectins, algal sulfated polysaccharides likesulfated fucans and galactans, agars, carrageenans, fucoidans, gluco-and galactomannans, arabinogalactans, glycosaminoglycans like hyaluronicacid, dermatan-sulfate, keratan-sulfate and degradation productsthereof, chondroitin-4-sulfate, chondroitin-6-sulfate, heparan-sulfate,heparin and derivatives thereof, pentosans, dextrans, chitosan andderivatives thereof, hydrosoluble and hydrodispersible derivatives ofstarch or cellulose like starch or cellulose alkylethers,hydroxyalkylethers or carboxyethylethers, like carboxymethylstarches,hydroxyethylstarches, carboxymethylcelluloses, and mixtures thereof.

Advantageously, the polysaccharide of the particle according to theinvention possesses an affinity for the growth factor of the particle,and is preferably negatively-charged.

The polysaccharide of the particle according to the invention is moreadvantageously selected from the group consisting of gum Arabic, alginicacid and derivatives thereof, alginic salts, alginic esters, algalsulfated polysaccharides like sulfated fucans and galactans, agars,carrageenans, fucoidans, glycosaminoglycans like hyaluronic acid anddegradation products thereof, chondroitin-4-sulfate,chondroitin-6-sulfate, dermatan-sulfate, keratan-sulfate,heparan-sulfate, heparin and derivatives thereof, and mixtures thereof.

Gum Arabic is also called acacia gum.

Alginic acid derivatives are preferably salts of alginic acid, such assalts of monovalent cation like sodium, lithium or potassium, preferablysodium.

Other preferred alginic acid derivatives are esters of alginic acid,where some of the carboxyl groups are esterified with a hydroxylcontaining compound, such as an alcohol or glycol, like ethylene glycol,propylene glycol, glycerol, preferably propylene glycol.

According to one embodiment, the membrane of the particles according tothe invention consists in a covalently cross-linked polysaccharide, onwhich at least one growth factor is adsorbed.

According to one embodiment, the polysaccharide of the particleaccording to the invention is chosen from the group consisting inalginic acid derivatives, gum Arabic, carrageenans, andchondroitin-sulfates.

According to one embodiment, the particle according to the inventionfurther comprises a protein which is co-cross-linked with thepolysaccharide.

As used herein, the term “co-cross-linked” refers to a protein bound tothe polysaccharide polymer, preferably by covalent bonds. Thecross-linking of the protein may occur after, or during thecross-linking of the polysaccharide molecules.

According to another embodiment, a particle according to the inventionpresents a core/membrane structure, wherein:

-   -   the membrane consists in a covalently cross-linked        polysaccharide co-cross-linked with a protein, on which at least        one growth factor is adsorbed, and    -   the core is liquid.

Said protein is preferably chosen from the group consisting of albuminslike serumalbumin, ovalbumin or alpha-lactalbumin, globulins,solubilized scleroproteins, collagen, atelocollagen, gelatin, elastin,hemoglobin, fibrinogen, fibrin, silk fibroin, milk proteins, casein,glycoproteins like fibronectin or mucin, plant proteins extracted from aleguminous or proteagenous plant, plant proteins extracted from acereal, and mixtures thereof.

According to one embodiment, the protein of the particle according tothe invention is an albumin-type protein, such as serum-albumin, and isfor example human serum albumin (HSA).

As used herein, the term “growth factor” refers to a naturally occurringsubstance capable of stimulating cellular growth, cellularproliferation, cellular differentiation and cellular maturation, suchas, for example, bone cell differentiation or blood vesseldifferentiation.

According to one embodiment, the growth factor is adsorbed to thecovalently cross-linked polysaccharide comprised in the membrane or thewhole volume of the particle according to the invention.

Advantageously, said covalently cross-linked polysaccharide forms anetwork and the growth factor is bound to the said network, preferablyby electrostatic interactions.

According to another embodiment, the membrane of the particles accordingto the invention consists in a covalently cross-linked polysaccharide,co-cross-linked with a protein, on which at least one growth factor isadsorbed.

A particle according to this embodiment comprises propylene glycolalginate as cross-linked polysaccharide, and human serum albumin asprotein co-cross-linked with the polysaccharide.

Another particle according to this embodiment comprises covalentlycross-linked gum Arabic, co-cross-linked with human serum albumin.

Another particle according to this embodiment comprises covalentlycross-linked chondroitin-sulfate, co-cross-linked with human serumalbumin.

Another particle according to this embodiment comprises propylene glycolalginate and sodium alginate as cross-linked polysaccharides, and humanserum albumin as protein co-cross-linked with the polysaccharides.

According to one embodiment, the particle according to the inventioncomprises at least two growth factors.

The particles according to this embodiment preferably comprise twodifferent growth factors.

Such particles enable the controlled spatiotemporal delivery ofdifferent growth factors, which induce different therapeutic effectsaccording to different kinetics of release. It has been observed thatsuch particles synergistically enhance the therapeutic effects of eachgrowth factor.

According to this embodiment, it is preferable to select the nature ofthe particles in order to adjust the kinetics of release of each growthfactor.

According to this embodiment, it is also preferable to associate on thesame particle, two growth factors with shifted kinetics of release.

According to one embodiment, the membrane of the particles according tothe invention consists in a covalently cross-linked polysaccharide, onwhich at least two growth factors are adsorbed.

According to this embodiment, the membrane of the particles consists incovalently cross-linked propylene glycol alginate, gum Arabic,carrageenan, or chondroitin-sulfate.

According to another embodiment, the membrane of the particles accordingto the invention consists in a covalently cross-linked polysaccharide,co-cross-linked with a protein, on which at least two growth factors areadsorbed.

A particle according to this embodiment comprises propylene glycolalginate as cross-linked polysaccharide, and human serum albumin asprotein co-cross-linked with the polysaccharide.

Another particle according to this embodiment comprises covalentlycross-linked gum Arabic, co-cross-linked with human serum albumin.

Another particle according to this embodiment comprises covalentlycross-linked chondroitin-sulfate, co-cross-linked with human serumalbumin.

Another particle according to this embodiment comprises propylene glycolalginate and sodium alginate as cross-linked polysaccharides, and humanserum albumin as protein co-cross-linked with the polysaccharides.

In the particles according to the present invention, the species thatform the membrane (polysaccharides and eventual proteins) areessentially covalently cross-linked, thus leaving the negatively-chargedsites of the membrane fully available to interact with the growthfactors, notably with their positively-charged sites.

On the contrary, in an ionically cross-linked particle, most of thepolarized sites of the membrane are already engaged in an ionic bondwith another site of opposite polarity. Those sites are thus lessavailable and such particles exhibit a weaker affinity with growthfactors.

Furthermore, proteins comprise various functional groups able to bindmolecules through ionic or hydrophobic interactions. The presence of aco-cross-linked protein in the membrane of the particles of theinvention brings additional binding sites for the growth factors,allowing a modulation of their release.

The growth factor of the particle according to the invention is chosenfrom the group consisting of fibroblast growth factors (FGFs),hepatocyte growth factor (HGF), platelet derived growth factors (PDGFs),vascular endothelial growth factors (VEGFs), angiopoietins (Angs),granulocyte colony-stimulating factor (GCSF), transforming growthfactors (TGFs), placental growth factors (PlGFs), epidermal growthfactor (EGF), stromal derived growth factor (SDF-1), insulin-like growthfactors (IGFs), nerve growth factor (NGF), osteogenin, or hormones suchas leptin, growth hormone (GH), estrogen, or cytokines such asinterleukins 1, 6, or 8, and mixtures thereof.

In one embodiment, the growth factor of the particle according to theinvention is preferably chosen from the group consisting of FGF-2, HGF,PDGF-BB and VEGF-A.

In one embodiment, the particle according to the invention comprises twodifferent growth factors. Preferably, the particle according to theinvention comprises HGF and FGF-2 as growth factors. Preferably, theparticle according to the invention comprises PDGF-BB and FGF-2 asgrowth factors.

In another embodiment, the particle according to the invention comprisesmore than two different growth factors.

The particle according to the invention is a growth factor deliverysystem capable of delivering growth factor combinations with differentkinetics, producing synergistic effects.

Within the present invention, it was found that polysaccharides aresuitable for the delivery of growth factor(s).

Within the present invention, it was found that alginate, a naturallyoccurring polysaccharide, is particularly suitable for the delivery ofpositively charged proteins, such as FGF-2, HGF, VEGF-A and PDGF-BB, asit bears negatively charged carboxylic groups available forelectrostatic interactions with the positively charged growth factors,thus slowing their release.

In addition, it was found that human serum albumin, a biocompatible andbiodegradable protein, brings enhanced mechanical resistance to theparticle network, and influences the release kinetics of the growthfactors.

Indeed, ionically cross-linked alginate hydrogels have been widely usedfor angiogenic growth factor delivery (Ruvinov et al. Biomaterials 31,4573 (2010); Hao et al. Cardiovascular Research 75, 178 (2007)), butgenerally display uncontrolled degradation leading to unpredictablerelease kinetics.

In contrast, the particles of the invention comprising covalentlycross-linked polysaccharide and particles further comprising proteinsco-cross-linked to the polysaccharide prevent hydrolysis-drivendissolution and delay proteases-driven degradation resulting in morestable particles with reproducible drug release rates.

Particles composed of cross-linked anionic polysaccharides, such asalginate, and eventually co-cross-linked proteins, such as HSA, thus canbe used to control the release of many different growth factors.Furthermore, the association of polysaccharides to proteins in across-linked network delays the degradation of the particles byproteases, which is very useful for the encapsulation of growth factorswith short half-lives.

Another particle according to the invention compriseschondroitin-sulfate as cross-linked polysaccharide, and human serumalbumin as protein co-cross-linked with the polysaccharide.

This natural glycosaminoglycan (chondroitin-sulfate) is likely to bindmore specifically heparin-binding growth factors, and allows theencapsulation of more growth factors for an even longer period ofdelivery.

The particles according to the invention are stable, biocompatible andbiodegradable.

Because of the strong covalent bonds maintaining their membrane, thecovalently cross-linked particles according to the invention are morestable than the ionically cross-linked particles of prior art.

The particles according to the invention are injectable, allowingspatiotemporal control over growth factor levels, and represent a lessinvasive delivery system than implantable delivery systems.

They are also a more potent delivery system for growth factors, and aretherefore of clinical interest, for example for the treatment ofcardiovascular disease patients, for the treatment of degenerativediseases, for the treatment of skin conditions related to aging, or fortissue engineering of in vitro cell culture.

The particles according to the invention are discrete particles, whichcan disperse independently into the tissue during their administration,leading to favourable concentration gradients, and avoiding the problemsassociated with too high local doses, including angioma formation oraberrant dysfunctional blood vessels.

Indeed, it was discovered that it is not the total amount of growthfactor(s) that determines the results of treatment, but rather thatlocal, microenvironmental concentration gradients play an important role(Ozawa et al, J. Clin. Invest. 2004, 113(4), 516-527).

The particles according to the invention allow better tissuedistribution of the growth factors around the site of injection.

The particles according to the invention also prevent undesiredwide-spread distribution of growth factors inside the target organ orinto the circulation.

Their slow-release property also allows the reduction in the requireddose to reach a certain biological effect, by enhancing the half-life ofthe growth factors and by protecting them from proteolysis. This resultsin reducing costs for the treatment but also importantly leads to asignificantly reduced risk of serious side effects from the treatment.

In a further aspect, the invention is directed to a method ofpreparation of the particles according to the invention, comprising astep of covalently cross-linking a polysaccharide, in order to provideparticles containing at least one covalently cross-linkedpolysaccharide.

The step of cross-linking of the method according to the invention istypically carried out by a method chosen from the group comprisinginterfacial cross-linking using acid dichlorides or acid dianhydrides,cross-linking using a transacylation reaction between a polysaccharidicester and a protein or a polysaccharide, cross-linking by aldehydes orpolyaldehydes, diisocyanates, diacrylates, carbodiimides, trisodiumtrimetaphosphate, diglycidylethers, epichlorohydrin, radiationcross-linking induced by electron beam or gamma rays exposure,photocross-linking, enzymatic cross-linking.

Alternatively, the step of cross-linking may be carried out by any othermethod allowing the formation of covalent bonds between thepolysaccharide molecules, at least on the surface of an aqueous droplet,in order to provide particles containing at least one covalentlycross-linked polysaccharide.

A first method for the step of cross-linking is interfacialcross-linking using acid dichlorides like terephthaloyl chloride,sebacoyl chloride, succinyl chloride, glutaryl chloride or adipoylchloride.

The following protocol illustrates this first method.

An aqueous solution of the polysaccharide, or mixture of polysaccharide,is divided in small droplets, by emulsification in a hydrophobic liquidadded with surfactant. A solution of the acid dichloride in the samehydrophobic liquid is then added to the emulsion. The acid dichloridediffuses through the emulsion and binds the functional groups of thepolysaccharide at the surface of the aqueous droplets through anacylation reaction, leading to the appearance of cross-links betweenpolysaccharides when the binding of the bifunctional cross-linking agentoccurs on two different polysaccharide molecules. The acylation reactionmay concern hydroxyl groups, amino groups, carboxylic acid groups, ofthe polysaccharide, leading to the formation of ester bonds, amidebonds, and anhydride bonds, respectively. A membrane made ofcross-linked polymer is progressively formed around each aqueousdroplet, leading to the individualization of microcapsules. The reactionis stopped by diluting the emulsion with organic solvent. The particlesare separated from the reaction medium by centrifugation, and subjectedto a series of washings in order to eliminate solvent and surfactantresidues.

Another method for the step of cross-linking is cross-linking using atransacylation reaction between a polysaccharidic ester like propyleneglycol alginate or pectin and a protein or a polysaccharide.

The following protocol illustrates this other method.

In this method, an aqueous solution containing protein, for examplealbumin, and polysaccharide, for example propylene glycol alginate(PGA), is divided in small droplets, by emulsification in a hydrophobicliquid added with surfactant. A solution of an alkaline agent likediluted sodium hydroxide solution is then added to the emulsion. Thealkaline agent diffuses through the emulsion and starts thetransacylation reaction between ester groups of polysaccharide and aminogroups of the protein, leading to the appearance of amide cross-linksbetween the two molecules. A membrane made of cross-linkedpolysaccharide and protein is progressively formed around each aqueousdroplet, leading to the initial individualization of microcapsules. Whenthe reaction progresses towards the center of each aqueous droplet, thenetwork made of cross-linked protein and polysaccharide progresses up tothe center as well, leading to the formation of microspheres. Thereaction is stopped by adding an acidic solution to the emulsion. Theparticles are separated from the reaction medium by centrifugation, andsubjected to a series of washings in order to eliminate solvent andsurfactant residues.

According to one embodiment, the method of preparation of the particlesaccording to the invention further comprises a step of imbibition of theparticles containing at least one covalently cross-linkedpolysaccharide, with a solution containing at least one growth factor.

Said step of imbibition is preferably carried out after the step ofcross-linking.

As used herein, the terms “loaded particles” refer to particles obtainedafter the step of imbibition with a growth factor.

In the embodiment wherein the particles present a liquid core, thegrowth factors may be bound to the membrane of the loaded particles andmay also be present in the solution forming the liquid core of theloaded particles.

In another embodiment wherein the particles present a matrix structure,the growth factors may be bound to the network constituting the loadedparticles and may also be present in the solution filling the pores ofthe network constituting the loaded particles.

Lyophilized particles are loaded with one or more growth factors (forexample HGF, FGF-2, PDGF-BB, and VEGF-A) by imbibition, using solutionsof said growth factors, in order to provide loaded particles, saidsolutions containing one or more growth factors.

In one embodiment, two (or more) fractions of the particle batch areseparated and separately loaded with different growth factors beforebeing associated in a composition, particularly in an injectablepharmaceutical composition. This method allows a great versatility toprecisely adjust the proportions and the loading of the differentfractions, depending on the desired sequential release kinetics.

According to one embodiment, the method of preparation of the particlesaccording to the invention further comprises a step of lyophilization ofthe particles.

Said step of lyophilization of the particles is preferably carried outafter the step of cross-linking and before the step of imbibition.

The method of preparation of the particles is very versatile and askilled man in the art can adapt this method to the preparation ofparticles within a large size range and having various degrees ofcross-linking.

The following protocol illustrates the formation of particles by themethod of interfacial cross-linking.

A protein and a polysaccharide, for example human serum albumin andpropylene glycol alginate, are dissolved in an aqueous phase. Thisaqueous phase is emulsified by stirring in an organic phase containing asurfactant. Then, an organic solution of acid dichloride is added to theemulsion and the cross-linking reaction is allowed to develop.

The reaction is stopped by dilution of the reaction medium. Theparticles are separated from the organic phase by centrifugation, andwashed.

Finally, the particles are freeze-dried by lyophilisation.

Lyophilized particles are loaded with one or more growth factors (forexample HGF, FGF-2, PDGF-BB and/or VEGF) by imbibition, using solutionsof said growth factors, in order to provide loaded particles, saidsolutions containing at least one growth factor.

In one embodiment, said step of imbibition is separately conducted withtwo or more fractions of particles, using two or more solutions ofgrowth factors.

In a further aspect, the invention is directed to the use of theparticles according to the invention for the controlled spatiotemporaldelivery of at least one growth factor.

In another aspect, the invention is directed to the particles accordingto the invention for the delivery of at least one growth factor tocultured cells in vitro for tissue engineering.

In another aspect, the invention also relates to the particles accordingto the invention for their use for stimulating wound healing such as inischemic skin wounds, diabetic ulcers, gastric ulcers, or in burnwounds, or for tissue regeneration in vivo, like therapeuticangiogenesis, bone regeneration, nerve regeneration, muscleregeneration, regeneration of hepatic tissue, regeneration of hybridtissue apparatus like periodontium, or for stimulating transplant organgrafting.

In another aspect, the invention also relates to the particles accordingto the invention for their use for the treatment of cardiovasculardiseases like ischemic heart disease, chronic heart failure, limbischemia or cerebral ischemia.

In another aspect, the invention also relates to the particles accordingto the invention for their use for the treatment of lymphedema bystimulation of lymphangiogenesis.

In another aspect, the invention also relates to the particles accordingto the invention for their use for the treatment of degenerativediseases like osteoarthritis, osteonecrosis, osteoporosis orperiodontitis, or neurodegenerative diseases such as alzheimers.

In another aspect, the invention also relates to the particles accordingto the invention for their use for the treatment of skin changes relatedto aging.

In a further aspect, the invention is directed to a pharmaceuticalcomposition comprising particles according to the invention, inassociation with a pharmaceutically acceptable vehicle.

In a further aspect, the invention relates to a pharmaceuticalcomposition comprising n series of particles according to the invention,in association with a pharmaceutically acceptable vehicle, each of saidseries of particles being loaded with a different growth factor, n being2, 3, 4 or more.

According to one embodiment, the pharmaceutical composition of theinvention comprises a first series and a second series of particlesaccording to the invention, wherein the first series of particles isloaded with a first growth factor and the second series of particles isloaded with a second growth factor, wherein the second growth factor isdifferent from the first growth factor.

Such pharmaceutical composition enables the controlled spatiotemporaldelivery of different growth factors, which induce different therapeuticeffects according to different kinetics of release. It has been observedthat such pharmaceutical composition synergistically enhance thetherapeutic effects of each growth factor.

According to this embodiment, it is preferable to select the nature ofthe particles in order to adjust the kinetics of release of each growthfactor.

According to this embodiment, it is also preferable to associate in thesame pharmaceutical composition, two growth factors with shiftedkinetics of release.

FIGURES

FIG. 1 a represents the distribution of size of albumin-alginateparticles of Example 1.

Particles have a mean diameter of 100 μm as observed by granulometry.

FIG. 1 b represents in vitro growth factor release rate ofalbumin-alginate particles of Example 1, loaded with FGF-2 (triangle),HGF (black square), PDGF-BB (white square) or VEGF-A (black dots),evaluated every other day for 6 weeks. Amounts are given as ng growthfactor released per day per mg of particles.

FIG. 1 c represents in vitro growth factor release rate of VEGF-A byalbumin-gum Arabic particles of Example 2 (black square), carrageenanparticles of Example 3 (white triangle), chondroitin particles ofExample 3 (white dots) and albumin-chondroitin particles of Example 2(white square), loaded with VEGF-A. Amounts are given as ng growthfactor released per mg of particles per day.

FIG. 1 d represents in vitro growth factor release rate of HGF byalbumin-alginate particles of Example 1 (black square) or alginateparticles of Example 3 (white dots), loaded with HGF. Amounts are givenas ng growth factor released per mg of particles per day.

FIG. 1 e represents in vitro growth factor release of VEGF byalbumin-alginate particles of Example 1 prepared by interfacialcross-linking method (black square) or albumin-alginate particles ofExample 6 prepared by transacylation method (white dots), loaded withVEGF. Amounts are given as ng growth factor released per mg of particlesper day.

FIG. 2 represents the stimulation of vascular cell migration,synergistically induced by FGF-2 and HGF.

The migration of HMEC-C (FIG. 2 a), HME (FIG. 2 b), and SMC (FIG. 2 c)is assayed in Boyden chambers for 6 hours.

Cells, pretreated or not with FGF-2 or HGF, were stimulated or not withHGF or FGF-2.

FIG. 3 represents the stimulation of vascular cell proliferation,synergistically induced by FGF-2 and HGF.

FIG. 3 a represents the proliferation of FGF-2-pretreated HMEC-C cellsstimulated or not with HGF.

FIG. 3 b represents the proliferation of FGF-2-pretreated HME cellsstimulated or not with HGF.

FIG. 3 c represents the proliferation of HGF-pretreated SMCs, stimulatedor not with FGF-2.

FIG. 3 d represents the proliferation of HGF-pretreated HMEC-C cellsstimulated or not with FGF-2.

Proliferation data is presented as fold increase over initial cellnumbers (mean±SEM, n=6 per group). *P<0.05; **P<0.01; ***P<0.001.

FIG. 4 represents the in vivo angiogenesis and arteriogenesissynergistically induced by the delivery of FGF-2 and HGF viaalbumin-alginate particles of Example 1.

Vessel formation, induced by microcapsule delivery of FGF-2 and/or HGF,was analyzed in Matrigel plugs in mice by immunohistochemical doublelabeling for CD31 (FIG. 4 a) and αSMA (FIG. 4 b).

Vascular density (FIG. 4 a) and mature vessel density (FIG. 4 b),quantified at 10×, is reported as the number of CD31⁺ and SMA⁺ vesselsper mm², respectively. Plug vessel content, quantified at 2.5×, ispresented as percentage vascularized area to the total area of thesection (FIG. 4 c).

Data is presented as mean determinants (±SEM; n=5 mice per group).*P<0.05; **P<0.01; ***P<0.001.

EXAMPLES Preparation and Characterization of the Particles Example 1Preparation of Growth Factor-Loaded Albumin-Alginate Particles UsingInterfacial Cross-Linking Method

Albumin-alginate particles can be prepared according to the followingmethod of interfacial cross-linking.

4% (w/v) human serum albumin (HSA, LFB) and 2% (w/v) propylene glycolalginate (PGA, ISP) are dissolved in a phosphate buffer pH 7.4. Thisaqueous phase is emulsified in cyclohexane (SDF) containing 2% (w/v)sorbitan trioleate (Sigma), at a stirring speed of 2000 rpm. Then, a2.5% (w/v) solution of terephthaloyl chloride (Acros) in achloroform-cyclohexane (1:4 v/v) mixture is added to the emulsion andthe cross-linking reaction is allowed to develop for 30 min.

The reaction is stopped by dilution of the reaction medium. Theparticles are separated from the organic phase by centrifugation, andwashed successively with cyclohexane, with ethanol(Charbonneaux-Brabant) containing 2% (w/v) polysorbate (Sigma), with 95%(v/v) ethanol and finally thrice with pure water.

Diameter measurements are performed using laser diffraction (ParticleSizer LS200, Beckman-Coulter). After staining with methylene blue, theparticles can be observed with a light microscope (Olympus, BH-2)equipped with interferential phase contrast. SEM observations(JSM-5400LV, JEOL) can be made after alcohol dehydration of particlesuspension followed by Au/Pd coating. Finally, the particles arefreeze-dried by lyophilisation in a Freezone 6 (LabConco, condensertemperature: −45° C., pressure <0.5 mbar).

Lyophilized particles are loaded with growth factors (HGF, FGF-2 and/orPDGF-BB) by imbibition, using 0.5-2 μg growth factor per mg particles(approximately 35,000 particles), during a 1 h-1 h30 incubation at +4°C.

The particles of Example 1 contain a thin, covalently cross-linked humanserum albumin (HSA) and propylene glycol alginate (PGA) membranesurrounding a liquid center. Laser diffraction measurements andmicroscopic observations revealed that these albumin-alginate particleshad a mean diameter of 100 μm and were roughly spherical (FIG. 1 a).Dehydration caused a partial and reversible collapse, resulting in theappearance of a very pleated surface in desiccated particles as observedby scanning electron microscopy.

Example 2 Preparation of Particles by Interfacial Cross-Linking ofVarious Polysaccharides Associated to Human Serum Albumin

The procedure described in Example 1 can be used to prepare particlesfrom the association of human serum albumin (HSA) to otherpolysaccharides using an interfacial crosslinking procedure.

The preparation parameters which differ from those of Example 1 arepresented in the table below.

Terephthaloyl chloride concentration in the chloroform-cyclohexanePreparation of the aqueous solution (1:4 v/v) mixture Sodium alginate(Sigma, 2% m/V) and 2.5% m/V HSA (LFB, 4% m/V) in Phosphate buffer, pH7.4 Gum Arabic (Spraygum AB, CNI, 20% 2.5% m/V m/V) and HSA (4% m/V) inPhosphate buffer, pH 7.4 Chondroïtin sulfate (Sigma, 10% m/V)   5% m/Vand HSA (4% m/V) in NaOH 0.1N

The particles are then washed, lyophilized and loaded with growthfactors as described in Example 1.

Laser diffraction measurements and microscopic observations revealedthat the particles of sodium alginate/HSA, gum Arabic/HSA, andchondroïtin sulfate had a mean diameter (+/−SD) of 86.02 (+/−44.34) μm,68.38 (+/−20.92) μm, and 101.9 (+/−38.91) μm respectively, and wereroughly spherical.

Example 3 Preparation of Particles by Interfacial Cross-Linking ofVarious Polysaccharides without the Addition of a Protein

The procedure described in Example 1 can be used to prepare particlesfrom various polysaccharides using an interfacial cross-linkingprocedure without the addition of a protein.

The preparation parameters which differ from those of example 1 arepresented in the table below.

Terephthaloyl chloride Emulsification step: concentration in thestirring speed and chloroform-cyclohexane Preparation of the aqueoussolution organic phase (1:4 v/v) mixture Propylene glycol alginate(Profoam, 4000 rpm in 2.5% m/V FMC Biopolymer) 5% m/V Cyclohexane + 1%in NaOH 1N (m/V) sorbitan trioleate Sodium alginate (Sigma) 3% m/V 4000rpm in 2.5% m/V in NaOH 1N Cyclohexane + 1% (m/V) sorbitan trioleate GumArabic (Spraygum AB, CNI) 1500 rpm in 2.5% m/V 10% m/V in NaOH 1NCyclohexane + 2% (m/V) sorbitan trioleate Carrageenan (Sigma) 2% m/V2800 rpm in 2.5% m/V in NaOH 1N at 60° C. Cyclohexane + 2% (m/V)sorbitan trioleate Chondroïtin sulfate (Sigma) 10% 3500 rpm in   5% m/Vm/V in NaOH 1N Cyclohexane + 2% (m/V) sorbitan trioleate

The particles are then washed, lyophilized and loaded with growthfactors as described in Example 1.

Laser diffraction measurements and microscopic observations revealedthat the particles of propylene glycol alginate, gum Arabic, andchondroïtin sulfate had a mean diameter (+/−SD) of 73.48 (+/−42.38) μm,63.86 (+/−35.21) μm, and 101.3 (+/−69.26) μm respectively, and wereroughly spherical.

Example 4 Preparation of Growth Factor-Loaded Albumin-Alginate ParticlesUsing the Transacylation Method

The aqueous phase is prepared by dissolving 2% (m/V) PGA (Profoam, FMCBiopolymer) and 20% (m/V) HSA (LFB) in distilled water. 6 mL of thisaqueous solution are then emulsified in 40 mL of isopropyl myristate(SDF) supplemented with 5% (m/V) sorbitan trioleate (Sigma) at astirring speed of 3000 rpm. After 5 minutes of stirring, 2 mL of a 2%(MN) solution of sodium hydroxide in ethanol 95% (V/V) (CharbonneauxBrabant) are added to the emulsion. The emulsion is neutralized after 15minutes by adding 2 mL of an 8.5 (V/V) solution of acetic acid inethanol 95% (V/V). The agitation is stopped after 15 minutes. Theparticles are then separated from the reaction medium by centrifugation,and the pellet is then resuspended in a 2% (m/V) aqueous solution ofpolysorbate (Seppic) and thrice in pure water.

The particles are then lyophilized and loaded with growth factors asdescribed in Example 1.

Laser diffraction measurements and microscopic observations revealedthat the particles of propylene glycol alginate/HSA had a mean diameter(+/−SD) of 92.23 (+/−47.36) μm and were roughly spherical.

Example 5 Preparation of a Composition Containing Two Fractions ofParticles Separately Loaded with Two Growth Factors for SequentialProlonged Release In Vivo

Growth factors stock solutions (about 100 ng/μl) are prepared assuggested by the manufacturer in PBS or H₂O depending on the specificgrowth factor. Stock solutions are stored at −80° C. until usage. Adefined amount of particles in an Eppendorf tube are loaded with onegrowth factor at the dose of 1 μg growth factor per mg particles(corresponding to about 35 000 particles). The total volume for theloading is kept at about 15-20 μL per mg particles (adjusted by additionof the release buffer, RB). The particles are incubated on ice for 1.5h. Release buffer is then added to prepare a suspension for injection orfor analyses of growth factor release.

For in vivo administration the following suspensions were prepared:

-   FGF-2 alone: ratio polymer/release buffer mg/mL 7,246-   HGF alone: ratio polymer/release buffer mg/mL 1,812

In each case, 23 μL of this solution were injected in three spots of theleft ventricle of rats making the total dose per heart 500 ng FGF2 or125 ng HGF.

-   FGF-2 for combination: ratio polymer/release buffer mg/mL 10,417-   HGF for combination: ratio polymer/release buffer mg/mL 5,952

16 μL of FGF-2-loaded particle suspension were mixed with 7 μL of theHGF-loaded particle suspension, and 23 μL of this mixed suspension wasinjected in three spots of the left ventricle making the total dose perheart 500 ng FGF-2+125 ng HGF.

For studies in mice, particles were charged at 1 or 2 μg growth factorper mg particles and the following solutions were prepared for injectionin the in vivo matrigel model:

-   PDGF-BB alone: ratio polymer/release buffer mg/mL 10.0-   VEGF-A alone: ratio polymer/release buffer mg/mL 10.0-   HGF for combination: ratio polymer/release buffer mg/mL 6.897-   FGF-2 for combination: ratio polymer/release buffer mg/mL 6.897

31 μL of FGF-2-loaded particle suspension were mixed with 8 μL of theHGF-loaded particle suspension, and 40 μL of this mixed suspension wasinjected in four spots in the left ventricle making the total dose perheart 500 ng FGF-2 or 125 ng HGF.

Example 6 Growth Factor Release Kinetics from Various Particles

The particles of Example 1 sequentially release FGF-2, HGF, PDGF-BB, orVEGF-A during more than 1 month, significantly increasing theirangiogenic effects in vivo.

In order to achieve spatiotemporally-controlled release of angiogenicgrowth factors, injectable, particulate delivery systems are used.

The growth factor-loaded particles are resuspended at 4 mg particles/mLin extracellular fluid mimetic release buffer (EFM-RB; 5 mM KCl, 125 mMNaCl, 20 mM Hepes, 1.5 mM MgCl2, 1.5 mM CaCl2, pH 7.4) and incubatedunder continuous rotation (6 rpm) for 40 days at 37° C. Every other daythe tubes are centrifuged (300 g, 8 min) to pellet the particles. Asample of the supernatant is collected and stored at −80° C. The initialvolume in the test tube is restored by addition of fresh EFM-RB tosimulate unlimited diffusion conditions. The growth factor-release isquantified by ELISA according to the manufacturer's instructions(VEGF-A, HGF and PDGF-BB, RnD systems; FGF-2, Invitrogen). Data arepresented as mean amount (ng) of growth factor released per day per mgof particles (n=3).

The particles of Example 1 were assayed in vitro for the release ofangiogenic growth factors under conditions approximating in vivo tissueenvironment. Whereas FGF-2 and VEGF-A release from the particles beganimmediately, the release of HGF and platelet-derived growth factor(PDGF)-BB were delayed for more than 1 week (FIG. 1 b). Further, whereasPDGF-BB release lasted 4 weeks, that of FGF-2, HGF and VEGF-A lasted 6weeks. In addition, it was confirmed that the growth factors releasedfrom the particles retained their full bioactivity using an in vitroassay of cell migration.

The particles of Examples 2 and 3 were also assayed in vitro for therelease of angiogenic growth factors under conditions approximating invivo tissue environment.

FIG. 1 c shows that the particles of polysaccharide co-crosslinked withHSA exhibit a first level of release for the first two weeks, higherthan the particles of polysaccharides crosslinked without protein.

FIG. 1 d shows that alginate particles, co-crosslinked with HSA or not,exhibit similar release rates of HGF, though the release rate ofalginate-albumin particles is slightly superior but begins few dayslater than alginate particles.

FIG. 1 e shows that the crosslinking method impacts the release rate ofgrowth factor. Interfacial crosslinking method enables a slow butconstant release while transacylation crosslinking method enables a fastbut decreasing release.

Example 7 Confocal Imaging of Growth Factor Distribution

To determine the growth factor localization, confocal analyses ofparticles loaded with fluorescently labeled FGF-2 or HGF were performed.

Twenty μg of rhFGF-2 or rmHGF were fluorescently labeled using anAlexa-555 kit according to the manufacturer's instructions (Microscaleprotein labeling kit, Invitrogen). Lyophilised particles prepared asdescribed in example 1 were loaded with the fluorescent growth factors,using 1 μg growth factor per mg particles as above. The growthfactor-loaded particles were resuspended at 4 mg particles/mL inextracellular fluid mimetic release buffer (EFM-RB, as above). Followingover night incubation, the particles were embedded in matrigel, andimaged using a Leica SP5 TCS X inverted confocal microscope at 20×.Images were processed using Leica LAS AF software (version 2.2.0). Theresults show that both growth factors bound to the microcapsule surfacelayer, confirming their interactions with the cross-linkedprotein-polysaccharide membrane. However, FGF-2 was also present in theliquid center of the microcapsule. These findings may in part explainwhy the particles display different release profiles for FGF-2 and HGF.

Biological Results Example 8 FGF-2 and HGF Synergistically StimulateVascular Cell Migration and Proliferation

Cell migration and proliferation: Chemotaxis can be assayed using amodified Boyden migration 48-well chamber (AP48; Neuro Probe Inc.,Gaithersburg, USA).

Briefly, membranes are coated with 0.15% gelatin for 1 h at 37° C.Cells, serum starved for 24 h in 1% FCS (without MV bullet kitsupplements for HMEC-C and PmT-EC), are stimulated with growth factorfor 24 h in the case of pre-treatment using FGF-2 (25 ng/ml for HME, PmTand RAOSMC; 50 ng/ml for HMEC-C) or HGF (25 ng/ml for HMEC-C; 50 ng/mlfor HME, PmT and RAOSMC). Next, cells are trypsinized and resuspended infresh medium supplemented with 0.25% BSA. Into each upper well was added1×10⁴ cells. The lower wells contained FGF-2 (12.5 ng/ml for HME andRAOSMC; 25 ng/ml for PmT-EC and HMEC-C) or HGF (12.5 ng/ml for HME,PmT-EC, and RAOSMC; 5 ng/ml for HMEC-C). The cells are incubated in thechamber for 6-12 h hours at 37° C., after which cells attached to themembrane are fixed in methanol and stained with Hematoxylin. Sixreplicate samples are used in each experiment, and experiments areperformed at least twice. Migrating cells are analyzed using a lightmicroscope, and reported as numbers of migrating cells per optic field.For analyses of cell proliferation, HME, HMEC-C, and RAOSMCs are eitherpretreated or not for 24 h with FGF-2 (25 ng/ml for HME; 50 ng/ml forHMEC-C and RAOSMCs) or HGF (25 ng/ml for HMEC-C; 50 ng/ml for HME andRAOSMCs). Next, 1×10⁴ cells are added to each well of 24-well plates andincubated for 2 h at 37° C. After cell attachment, the medium isreplaced with fresh medium containing FGF-2 (25 ng/ml for HME; 10 ng/mlfor HMEC-C; 50 ng/ml for RAOSMCs) or HGF (1 ng/ml for HME; 25 ng/ml forHMEC-C; 50 ng/ml for RAOSMCs). Cell proliferation is assayed after 24 h,48 h, or 72 h using the WST-1 assay according to manufacturer'sinstructions (Roche).

First, it is found that FGF-2 (Fibroblast Growth Factor-2) and HGF(Hepatocyte Growth Factor) synergistically stimulate vascular cellmigration and proliferation in vitro, and angiogenesis in vivo.

The effects of FGF-2 and/or HGF are evaluated in vitro using murineheart microvascular endothelial cells (HME), murine embryonicmicrovascular endothelial cells (PmT-EC), human microvascular cardiacendothelial cells (HMEC-C), and rat aortic smooth muscle cells (SMCs).

Whereas both FGF-2 and HGF used alone induced endothelial cell (EC) andSMC migration (FIG. 2) and proliferation (FIG. 3), HGF pretreatmentsignificantly potentiated EC motility response to FGF-2 (FIG. 2 a, 2 b).

Correspondingly, pretreatment with FGF-2 enhanced HGF-induced migration(FIG. 2 a, 2 b). Similarly, in SMCs, FGF-2 strikingly increased motilityresponses to HGF, although HGF did not alter FGF-2-induced migration(FIG. 2 c).

Moreover, pretreatment of HMEC-C (FIG. 3 a) or HME (FIG. 3 b) with FGF-2significantly increased HGF-induced cell proliferation. Conversely,pretreatment of HMEC-C (FIG. 3 d) or SMCs (FIG. 3 c) with HGFsignificantly increased cell proliferation responses to FGF-2.

These data show that FGF-2 and HGF synergistically stimulate vascularcell migration and proliferation, indicating that the combination ofthese growth factors may be useful for therapeutic angiogenesis.

Example 9 Effect of Growth Factor Loaded Particles on Angiogenic andArteriogenic Responses In Vivo (Mouse Matrigel Plug Model)

Growth factor-reduced matrigel (BD, Bedford, Mass., USA) can be used toevaluate angiogenic responses in vivo. Briefly, 500 μL matrigel is mixedwith 0.125, 0.5, or 1 mg particles of example 1 loaded with growthfactors as above. Controls contained matrigel mixed with buffer orparticles without growth factors. The matrigel mixture is subcutaneouslyinjected to form dorsal plugs in male Balb/c mice anesthetized byintraperitoneal injection of ketamine (90 mg/kg, Bayer, France) andxylazine (3.6 mg/kg). After 3 weeks, matrigel plugs are harvested,snap-frozen embedded in Tissue-Tek (Sakura Finetek, Torrance, USA) andstored at −80° C.

Semi-quantitative fluorescence microscopy: Angiogenic growth factorreceptor protein expression levels were determined in small and largerblood vessels in histological sections from matrigel plugs implantedwith particles loaded with either FGF-2 or HGF, as above. Briefly, 10 μmthick acetone-fixed cryosections were double labeled for either c-Met(rabbit antimouse c-Met, 1:300, sc162, Santa Cruz Biotech, USA) orFGFR-1 (rabbit anti human FGFR1, 1:100, Sigma), in conjunction with CD31(biotinylated rat antimouse CD31, 1:100, BD). Secondary reagentsincluded a donkey antirabbit-Cy3 (1:300, Jackson ImmunoresearchLaboratories) and SA-FITC (1:200, Sigma). Following counterstaining withHoescht's dye (1:10,000) and mounting in Vectashield, the sections wereviewed using a Leica SP5 TCS X inverted confocal microscope at 40×(NA=1.25) and images were acquired in a sequential mode. Confocalsettings were kept strictly identical between series. In addition;homogeneity of field illumination and power laser output was verifiedprior to and after experiments to ensure stable conditions ofacquisition. Images were analyzed using Bitplane Imaris software(version 6.10) to determine maximal relative levels of signal intensityin regions of interest (ROI) centered on individual blood vessels. Intotal 3-5 animals per group, and for each animal 5-7 images, eachincluding 7-50 ROI, were analyzed.

To determine if the slow-release system of the invention would influencethe angiogenic effect of FGF-2 or HGF in vivo, the comparison oftreatment with growth factor-loaded particles versus naked growthfactors was studied using the mouse matrigel plug model. Growthfactor-delivery by particles was found to be 2-6 times more potent toinduce angiogenesis, as compared with bolus delivery of growth factors.

The reciprocal stimulatory interactions observed between FGF-2 and HGFin vascular cells suggested that these growth factors may cooperativelyregulate vessel growth. To investigate this possibility, particlescontaining FGF-2 and/or HGF were injected in matrigel plugs in mice. Ineach case, the lowest dose of growth factor resulting in a substantialangiogenic effect was employed. Whereas each growth factor used aloneinduced a moderate angiogenic response, FGF-2 and HGF used incombination synergistically stimulated angiogenesis and arteriogenesis,as evidenced by the increased vascular density (FIG. 4 a), vascularmaturity (FIG. 4 b), and vascularized area (FIG. 4 c) as compared tosingle growth factor treatments.

Similar results were obtained with particles containing PDGF-BB and/orFGF-2. Whereas each growth factor used alone induced a moderateangiogenic response, PDGF-BB and FGF-2 used in combinationsynergistically stimulated angiogenesis and arteriogenesis, as evidencedby an increased vascular density, vascular maturity, and vascularizedarea as compared to single growth factor treatments.

The most potent angiogenic growth factor combination described to dateis the association of FGF-2 and PDGF-BB (Cao et al Nat. Med. 2003, 9(5),604-613). To compare these two different growth factor combinations,particles containing FGF-2 and/or PDGF-BB were injected in matrigelplugs in mice. It was found that whereas the vessel density induced bythe combination of FGF-2 and PDGF-BB was moderately greater than thatinduced by FGF-2 in combination with HGF, the number of mature vesselsdid not differ.

However, whereas the combination of FGF-2 and PDGF-BB only resulted in30% of the total plug area being vascularized, the combination of FGF-2and HGF notably resulted in a matrigel plug vessel content of around80%.

These results reveal the albumin-alginate particles as particularlyefficient for growth factor delivery in vivo. Further, in agreement withthe in vitro data, the combination of FGF-2 and HGF synergisticallyinduced angiogenesis and arteriogenesis at a level comparable orsurpassing that of the most potent angiogenic growth factor combinationcurrently described.

Example 10 Effect of Growth Factor Loaded Particles afterIntramyocardial Injection

Myocardial infarction (MI) was induced in anesthetized (ketamine, 3.6mg/kg; isoflurane gas, 2%), mechanically ventilated male Wistar rats byligation of the proximal left coronary artery following a leftthoracotomy (n=22 rats/group) as previously described^(26, 28). Ratswith infarcts encompassing 20-50% of the left ventricle (LV) wereincluded in the study. Immediately after, particles loaded with growthfactors as outlined above were injected in three spots (23 μl/spot)along the infarct border zone on the right anterior side of the LV freewall adjacent to the septum. The total amount of growth factoradministered per heart was 125 ng HGF or 500 ng FGF-2 alone or thecombination of both. Controls were injected with the same numbers ofparticles without growth factor. The thorax was closed in three layersand rats allowed to recover on a heating pad before being returned totheir cages (2-3 rats/cage). At the time of sacrifice, rats were given alethal dose of anaesthesia (Sodium Methohexital) followed by rapidexcision of the heart through a ventral thoracotomy. Hearts werearrested in diastole by immersion in ice-cold saturated potassiumchloride buffer. Lungs and hearts were weighed and the LV dissectedbefore being cut transversally at the level of the papillary muscles andsnap-frozen embedded in Tissue-Tek and stored at −80° C.

Histochemistry: MI size and collagen density were determined in 10 μmthick serial heart cryosections fixed in Carnoy's fixative and stainedwith Sirius Red. Slides were examined and photographed under a lightmicroscope (Zeiss) at 40× magnification. Collagen-rich areas wereidentified using Image Pro Plus (version 6.3). Collagen content wascalculated as percentage of collagen area to total area of the image(n=14-16 animals/group). Infarct size was analyzed invideomicrophotographs of the sections using Adobe Photoshop (CS3extended version 10) to delineate epicardial and endocardial LVperimeters and infarct perimeter. Infarct size was calculated as: totalinfarction perimeter/(epicardial LV perimeter+endocardial LVperimeter)×100. For immunohistochemical analyses, 10 μm cryosectionswere post-fixed in acetone and stained according to standard protocolsusing rat antimouse CD31 (PECAM-1, 1:100, BD); biotinylated mouseantirat CD31 (PECAM-1, 1:100, BD); mouse antihuman SMA-FITC (smoothmuscle a actin, 1:200, Sigma); rabbit anti human Ki67 (1:500, NovocastraLaboratories, Newcastle, UK); and WGA-A488 (wheat germ agglutinin,1:100, Invitrogen). Secondary reagents included: streptavidin(SA)-Fluoprobe 547 (1:1500, Interchim, France), SA-Cy5 (1:1000, GEHealthcare Life Sciences, Uppsala, Sweden), donkey anti-rabbit Cy3(1:300, Jackson Immunoresearch Laboratories, Inc., West Grove, USA), orVectastain® ABC kit containing HRP-conjugated anti-rat IgG (VectorLaboratories, Burlingame, USA) used with DAB substrate kit (VectorLaboratories) for peroxidase staining. Sections were counterstained for2 min with Hoescht's dye (1:10,000, Sigma). Micrographs (n=2-4sections/animal, 5-15 animals/group) were captured using 10×, 20× or 40×objectives on a fluorescence microscope (Zeiss Axiolmager Z1) equippedwith an Apotome, or using a 2.5× objective on a light microscope(Leica). Images were processed by an operator blinded to the treatmentgroups using Image Pro-Plus, AxioVision (version 4.6), or AdobePhotoshop image analysis software. Vessel density and vessel maturitywere quantified as the number of CD31⁺ vessels and SMA⁺ vessels per mm²respectively. Matrigel plug vessel content was calculated as percentageof vascularized area to total section area. Endothelial proliferationwas analyzed by Ki67 and CD31 double labeling, and presented as numberof Ki67⁺ endothelial cells per mm². Cardiomyocyte sizes were measured inWGA-stained sections. Vessel to cardiomyocyte ratio was calculated asnumber of vessels to number of cardiomyocytes per mm².

MRI: Cardiac perfusion was assessed by arterial spin labeling MRI usinga 4.7T small animal magnet (Biospec 47/40 advanced II, Brucker,Ettlingen, Germany) equipped with a gradient insert (BGA12S 400 mT/m)and a transmit/recieve radiofrequency coil in quadrature mode with 86 mminternal diameter (Brucker).

Briefly, animals were anesthetized with methohexital and placed in asupine position on a warming cradle. ECG signal were monitored byplacing two subcutaneous electrodes on each side of the chest connectedto a small animal monitoring and gating system (1025-S-50 model, SAInstruments Inc., NY, USA) for MRI synchronization. After optimizationof the radio frequency signal, the perfusion sequence was run in theshort-axis plane allowing determination of myocardial tissue perfusion.Global as well as slice-selective spin inversion recovery T1* (fittedtime constant) maps were acquired. During acquisition 32 signal averageswere performed resulting in an imaging duration of about 20 min peranimal⁴⁷. Regional perfusion in the treated area of the LV wascalculated as P=((0.95/T1_(internal))×(T1*_(global)/T1*_(selective))−1)of the ROI where T1_(internal) is the signal in the blood⁴⁸. Perfusionimages were analyzed with ParaVision 5.0 software (Brucker) by twoindependent observers.

Echocardiography: Animals (n=15-17 rats/group, and 8 age-matchedsham-operated rats) were examined at 1 month and 3 months post-MI bytransthoracic echocardiography. 2D images and 2D-guided M-mode, DopplerM mode, and pulsed-wave Doppler recordings were obtained fromparasternal short axis (level of papillary muscles) views, using a Vivid7 Ultrasound (GE Healthcare) echograph with a M12L linear probe operatedat 14 MHz, and analyzed using Echopac PC software. LV end-diastolicdiameter (EDD) and end-systolic diameter (ESD) were measured by theleading-edge convention, and used to calculate fractional shortening(FS) through the equation FS=[(EDD−ESD)/EDD]×100. Velocity-time integral(VTI) was measured at the level of the pulmonary artery by pulse-waveDoppler. LV free wall AW end-diastolic thickness (AWT ED) and AWend-systolic thickness (AWT ES) were measured in TM mode captured inshort-axis views, and used to calculate LV Anterior Wall fractionalthickening (AW FT) by the equation AW FT=[(AWT ES−AWT ED)/AWT ED]×100.Rate-corrected Velocity of circumferential shortening (VCFc) wascalculated as VCFc=% FS/LVET×(R—R), where LVET=LV ejection time (ms) andR—R=ECG R—R interval. The mean of three consecutive cardiac cycles wasused for all measurements in each animal. Measurements, performed by asingle echocardiographer blinded to the treatment groups, were made inaccordance with the conventions of the American Society ofEchocardiography.

Results:

Intramyocardial delivery of FGF-2 and HGF stimulates angiogenesis andarteriogenesis and prevents MI-induced cardiac hypertrophy and fibrosis.

To evaluate the effect of FGF-2 in combination with HGF in a setting ofcardiovascular disease, a randomized, blinded experiment in ratssurviving coronary artery ligation (n=102) or sham surgery (n=11) wasperformed.

The experimental MI model leads to the development of CHF within 3months. Particles of Example 1, loaded or not with FGF-2 and/or HGF,were locally injected in the viable free wall bordering the leftventricular (LV) infarct zone immediately following MI.

At 1 or 3 months post-MI, the angiogenic and arteriogenic cardiaceffects were evaluated by immunohistochemistry.

At 1 month post-MI, untreated control rats displayed myocardial vesselrarefaction, including reduced levels of mature blood vessels, ascompared with healthy shams. Due to an inherent compensatory angiogenicresponse, evidenced by increased EC proliferation at 1 month, the totalvessel and mature vessel densities were slightly improved in untreatedcontrol hearts at 3 months. FGF-2 monotherapy resulted in a furtherincrease in angiogenesis and arteriogenesis locally in the treated LVarea, leading to slightly augmented vessel density and significantlyincreased mature vessel density at 1 month as compared with controls.However, the effects were lost at 3 months.

HGF monotherapy, on the other hand, showing limited arteriogenic effectsat 1 month, tended to increase EC proliferation and vascular density at3 months.

In contrast, the combination therapy induced a potent angiogenic andarteriogenic response, again strictly limited to the treated LV zone,with more than a doubling of the number of proliferating ECs and 3 timesmore mature blood vessels as compared with untreated controls at 1 monthpost-MI. Notably, by 3 months, the myocardial vessel density in thecombination group had attained normal sham rat levels. Moreover, themature vessel density in the group treated by FGF-2 and HGF incombination even surpassed that of shams.

Next, the extent of cardiac hypertrophy and fibrosis was evaluated byhistological analyses of cardiomyocyte sizes and collagen densityrespectively. Whereas FGF-2 treatment had no effect on these parameters,both HGF alone and the combination treatment reduced cardiac fibrosisand hypertrophy at 3 months. Notably, the coordinated decrease incardiomyocyte sizes and increase in blood vessel density generated bythe combination treatment resulted in a normalization of thecardiomyocyte:vessel ratio.

Furthermore, as it was observed that therapeutic effects were limited tothe injected LV zone, suggesting that the growth factors indeed had beensuccessfully, spatially targeted, the cardiac distribution of theslow-delivery vehicles was verified using fluorescently labeledparticles of Example 1. Hearts were evaluated histologically at 6 h, 1or 2 weeks after intramyocardial injection. The particles were found tobe strictly confined to a small area surrounding the three injectionpoints, extending maximally 1-2 mm into the subepicardial myocardium.The particles spread over an area representing around 8% of across-section the LV at the papillary muscle level after 6 h,progressively decreasing to around 6% after 1 week and 4.5% after 2weeks.

These findings confirm i) a localized microcapsule distribution centeredaround the points of injections in agreement with the observedrestricted LV effects of the therapies; ii) a comparable timeline ofmicrocapsule degradation in the heart as seen in vitro.

Intramyocardial delivery of FGF-2 and HGF improves regional cardiacperfusion and cardiac function following MI.

To assess if the blood vessels induced by the angiogenic therapiesresulted in a functional improvement in cardiac perfusion, magneticresonance imaging (MRI) was employed.

The results, obtained 3 months post-MI, demonstrate that whereasuntreated controls displayed significantly reduced cardiac perfusioncompared with healthy sham-operated animals, only the combinationtreatment increased regional cardiac perfusion in the treated area ofthe LV.

Next, to investigate whether the multiple beneficial myocardialalterations induced by the angiogenic therapies correlated with improvedcardiac function, even though myocardial infarct sizes were similar inall groups, echocardiographic analyses were carried out at 1 and 3months post-MI.

Untreated controls displayed severe cardiac dysfunction, characterizedby LV wall thinning at 1 month, and progressive LV dilation associatedwith the development of CHF. Both regional and global cardiaccontractility were reduced, as evidenced at 1 month by a decreasedfractional shortening (FS) and velocity of circumferential fibershortening (VCFc). HGF monotherapy only slightly improved LV parametersby 3 months, evidenced by decreased LVESD and end-diastolic and systolicvolumes and a tendency for increased FS and VCFc as compared withuntreated controls. FGF-2 monotherapy, on the other hand, significantlyreduced LV dilation, and by 3 months post-MI increased FS and VCFc.

In contrast to these moderate effects of monotherapies, the combinationof FGF-2 and HGF reduced LV dilation and LV dysfunction already at 1month post-MI, as shown by an increased FS and VCFc as compared withuntreated controls. This was associated with an increased LV anteriorend-systolic wall thickness, and a tendency for increased wallthickening. At 3 months, the combination treatment group displayed amajor increase in LV wall thickness and wall thickening as compared withuntreated controls and single growth factor groups. Moreover, the LVdilation was further reduced, and associated with a marked recovery ofLV function, evidenced by an increased FS and VCFc as compared with bothuntreated controls and HGF-treated animals, indicating that thedevelopment CHF was partially prevented.

Material & Methods

Reagents and Animals

Murine Heart Microvascular Endothelial cells (HME) were a kind gift fromDr Marco Presta (Università di Brescia, Italy). Murine EmbryonicMicrovascular Endothelial cells (PmT-EC) were a kind gift from Drs.Karin Aase and Lars Holmgren (Karolinska Institutet, Sweden). PrimaryHuman Microvascular Cardiac Endothelial cells (HMEC-C) and Rat AorticSmooth Muscle Cells (SMC) were purchased from Lonza. All cell lines weremaintained in DMEM (Gibco, Invitrogen, Paisley, UK) containing 10% FBS,except PmT-EC and HMEC-C, which were maintained in EGM-2 mediumsupplemented with MV bullet kit (Lonza). Primary cell cultures were usedfor experiments between passages 4-8. All cells were maintained at 5%CO₂ and 95% air at 37° C. Growth factors, rhFGF-2 (monomer, 157 a.a.),rmHGF (dimer, 463 and 232 a.a.), rhHGF (dimer, 697 a.a.), rhPDGF-BB(dimer, 109 a.a.), were obtained from RnD Systems Inc. (Minnesota, USA).Male Balb/c mice (20-22 g) and male Wistar rats (200-220 g) werepurchased from Janvier (Le Genest St Isle, France). Animal experimentswere performed in accordance with NIH guidelines, EU regulations, andFrench National legislation.

Statistics

Data are presented as mean determinants+SEM. Student's t-test(two-tailed) was used to compare two groups of independent samples. Formultiple comparisons, one-way analysis of variance (ANOVA) was employedusing GraphPad Prism software (version 5.0), followed by Tukey'spost-hoc test, except for echocardiographic data where repeatedmeasurements two-way ANOVA was employed followed by Bonferroni post-hoctest. A p<0.05 was considered significant.

The invention claimed is:
 1. A method of treatment of cardiovasculardiseases in a patient in need thereof, comprising administering to saidpatient a therapeutically effective amount of at least one growthfactor, wherein said at least one growth factor is released fromparticles containing a network or a membrane and said at least onegrowth factor, wherein said network or membrane comprises at least onecovalently cross-linked polysaccharide and a protein, wherein saidprotein is co-cross-linked with the at least one covalently cross-linkedpolysaccharide, wherein said at least one growth factor is adsorbed byelectrostatic interactions to the at least one covalently cross-linkedpolysaccharide, wherein the polysaccharide is selected from the groupconsisting of acacia gum, alginic acid and derivatives thereof, alginicsalts, alginic esters, chondroitin-4-sulfate, chondroitin-6-sulfate, andmixtures thereof, wherein the protein is selected from the groupconsisting of albumins, said therapeutically effective amount beingadministered by injection into a tissue of the patient, and wherein theparticles provide sustained release of the growth factor over a periodof at least 15 days.
 2. The method according to claim 1, wherein thegrowth factor is selected from the group consisting of fibroblast growthfactors (FGFs), hepatocyte growth factor (HGF), platelet derived growthfactors (PDGFs), vascular endothelial growth factors (VEGFs),angiopoietins (Angs), granulocyte colony-stimulating factor (GCSF),transforming growth factors (TGFs), placental growth factors (PIGFs),epidermal growth factor (EGF), stromal derived growth factor (SDF-1),insulin-like growth factors (IGFs), nerve growth factor (NGF),osteogenin, or hormones, and mixtures thereof.
 3. The method accordingto claim 1, wherein the at least one growth factor is at least twogrowth factors.
 4. The method according to claim 1, wherein a diameterof each of said particles ranges from 5 μm to 1,000 μm.
 5. The methodaccording to claim 1, wherein the particles have a core/membranestructure wherein: the membrane comprises the at least one covalentlycross-linked polysaccharide and the at least one growth factor adsorbedon the at least one covalently cross-linked polysaccharide, and the coreis liquid.
 6. The method according to claim 1, wherein the particleshave a matrix structure formed of a said network comprising the at leastone covalently cross-linked polysaccharide, the protein co-cross-linkedwith the at least one covalently cross-linked polysaccharide, and the atleast one growth factor adsorbed by electrostatic interactions on the atleast one covalently cross-linked polysaccharide, said network fillingthe whole volume of the particles and having pores wherein an aqueoussolution is entrapped.
 7. The method of claim 1, wherein said at leastone growth factor is adsorbed by electrostatic interactions to the atleast one covalently cross-linked polysaccharide at a surface of saidparticles.
 8. The method of claim 1, wherein said at least one growthfactor includes FGF2 and HGF.
 9. The method of claim 1, wherein saidstep of administering results in spatially targeted delivery of said atleast one growth factor to an injection zone.
 10. The method of claim 1,wherein said particle contains said network and said network is presentthroughout the whole volume of the particle.
 11. The method of claim 1,wherein said particle contains said membrane and said membrane surroundsa liquid core within said particle.
 12. The method of claim 1, whereinsaid particles are growth factor delivery systems delivering acombination of growth factor with different kinetics.
 13. The method ofclaim 12, wherein said combination of growth factors includes one ormore of FGF-2, HGF, PDGF-BB, and VEGF-A.